Sperm DNA fragmentation testing in clinical management of reproductive medicine

Abstract Background Approximately 8%–12% of couples worldwide face infertility, with infertility of individuals assigned male at birth (AMAB) contributing to at least 50% of cases. Conventional semen analysis commonly used to detect sperm abnormalities is insufficient, as 30% of AMAB patients experiencing infertility show normal results in this test. From a genetic perspective, the assessment of sperm DNA fragmentation (SDF) is important as a parameter of sperm quality. Methods In this narrative study, we review and discuss pathophysiological causes, DNA repair mechanisms, and management of high SDF. We then summarize literature exploring the association between SDF and reproductive outcomes. Main Findings Recent systematic reviews and meta‐analyses have revealed a significant association between high SDF in AMAB individuals and adverse reproductive outcomes including embryo development, natural conception, intrauterine insemination, and in vitro fertilization. However, the association with live birth rates and pregnancy rates following intracytoplasmic injection remains inconclusive. The disparities among quantitative assays, inconsistent reference range values, absent high‐quality prospective clinical trials, and clinical heterogeneity in AMAB patients with elevated SDF represent the main limitations affecting SDF testing. Conclusion The evaluation and management of SDF plays an important role in a subset of AMAB infertility, but widespread integration into clinical guidelines will require future high‐quality clinical trials and assay standardization.

ensuring transmission of a properly functioning genome to the next generation.The efficient production of healthy mature spermatozoa through spermatogenesis and spermiogenesis is a continuous and essential process for the fertility of individuals assigned male at birth (AMAB). 3comprehensive andrological consultation and examination are essential for all AMAB individuals seeking assistance for infertility.
The standard evaluation for infertility typically involves a semen analysis (SA), which assesses sperm production, motility, and viability.However, SA lacks the ability to determine the functional potential of sperm to fertilize an oocyte and contribute to embryonic development. 4While SA can determine abnormal semen conditions like asthenozoospermia, oligozoospermia, and azoospermia, this test cannot definitively determine fertility or infertility in all patients. 5,6e WHO has established reference values for SA parameters arbitrarily based on 5th percentile cutoffs from a large population from various countries.However, the predictive value of these reference values has been a subject of ongoing debate, as no single or set of semen parameters is highly indicative of AMAB fertility. 7,8Moreover, SA exhibits considerable variability over time, underscoring the need for at least two SA tests to ensure a thorough evaluation. 4Moreover, relying solely on reference semen analysis values to support natural conception is no longer applicable in the era of in vitro fertilization (IVF)/intracytoplasmic injection (ICSI).These assisted reproductive technologies (ART) require fewer but arguably higher-quality spermatozoa for successful pregnancies and live births. 8Idiopathic infertility is widely recognized as the primary cause of AMAB infertility, accounting for approximately 40%-50% of cases. 2 However, since up to 30% of infertile patients AMAB exhibit normal results in conventional SA, this method does not capture all relevant information and cannot be solely relied upon to detect sperm abnormalities. 9,10nsequently, there is a growing demand for objective parameters and seminal markers to improve infertility diagnosis.Some laboratories have incorporated advanced sperm tests, such as assessing acrosome functionality, chromatin condensation, or DNA fragmentation, into their andrological evaluations.
Sperm DNA fragmentation (SDF) is a parameter related to AMAB fertility that conventional SA fails to directly identify.The rationale behind SDF testing is to assess the genetic integrity of spermatozoa.SDF can occur due to defective chromatin condensation or protamination during spermiogenesis, unsuccessful apoptotic processes, and oxidative stress caused by imbalances in reactive oxygen species (ROS) and antioxidants. 11,12Numerous studies have demonstrated that infertile AMAB patients often have elevated SDF levels associated with various risk factors including smoking, drug use, and lifestyle choices. 13,14Recently, the WHO laboratory manual included SDF as an extended examination and provided detailed procedures for its assessment. 4Global guidelines now increasingly recommend measuring SDF in specific situations including recurrent pregnancy loss (RPL) and cases of "unexplained" AMAB infertility, especially when conventional SA yields normal results. 15,16spite this recommendation, the results of DNA fragmentation studies are not consistently reliable due to various factors.
These factors include the absence of standardized cutoff values to differentiate between fertile and infertile patients or to predict reproductive outcomes, non-standardized protocols measuring a mix of single and double strand breakages, the use of different testing assays that measure unrelated parameters for assessing DNA fragmentation, and the lack of randomized controlled trials (RCTs) assessing efficacy. 17Thus, the question of whether SDF measurement should be routinely incorporated into fertility evaluations remains a topic of debate.Nevertheless, SDF prevails as a potential objective biomarker for diagnosing and evaluating AMAB infertility and guiding treatment decisions. 18The purpose of this narrative review is to provide an updated summary of the current evidence concerning SDF measurement in clinical practice and its impact on clinical decision-making.In addition, we aim to explore the pathophysiological aspects of SDF, including its underlying causes, repair mechanisms, and strategies for managing high SDF.

| MATERIAL S AND ME THODS
This narrative review utilized the PubMed/MEDLINE database for conducting the literature search.The search strategy encompassed several key terms, such as "sperm DNA fragmentation/damage," "DNA fragmentation index," "male infertility," "spontaneous pregnancy," "reproductive outcome," "recurrent pregnancy loss," "sperm DNA repair," and "artificial reproductive technologies."The comprehensive literature search was conducted from February 1st 2023 to April 30th 2023.Articles were included in this review if they were written in the English language and if their titles or abstracts were relevant to the context of this review.Comments, editorials, and non-English articles were excluded from consideration.Additionally, relevant articles were manually identified from the reference lists of the included articles.

| Molecular mechanisms of SDF
The presence of sperm DNA damage in the ejaculate can be attributed to three different mechanisms: defective chromatin condensation or protamination during the last stage of spermatogenesis, abortive apoptotic processes, and oxidative stress resulting from imbalances in ROS and antioxidants. 11DNA damage manifests as structural changes including base alterations, single-strand breaks (SSBs), and double-strand breaks (DSBs) and are summarized in  [18][19][20] Following SSB detection by PARP1/2, various end-processing proteins including XRCC1 protein complexes such as PNKP, APE1, and APE2 are recruited through interaction with XRCC1 and poly (ADP-ribose).This recruitment leads to the restoration of 3′-hydroxyl (OH) termini breaks through PKNP phosphatase and kinase activity, converting 3′-P and 5′-OH termini breaks into 3′-OH and 5′-P termini.This allows for DNA repair synthesis by DNA polymerases and DNA-end joining by DNA ligases. 21Bs are severe DNA lesions that can arise from either exogenous sources such as chemotherapeutic drugs or endogenous sources (e.g.ROS, mechanical stress, and replication aberrations).In specific cellular processes like meiosis, intentional DSBs are generated by nucleases to initiate homologous chromosome recombination.DSBs are highly dangerous and can trigger apoptosis since their repair mechanisms are more challenging than other types of DNA damage; they cannot utilize a sister strand as a template for repair.
Rejoining the broken strands through ligation can result in carcinogenic mutations and translocations or even lead to tumorigenesis.
[24] The discovery of H2AX (gH2AX) as a potential biomarker for cellular responses to DSB has been crucial in understanding DNA damage response (DDR) mechanisms.When DSBs occur, phosphorylation of the Ser-139 residue of the histone variant H2AX by members of the phosphatidyl-inositol-3-kinase-related kinases (PIKKs) family takes place, resulting in the formation of gH2AX.This modification enhances DNA accessibility through chromatin modification, which leads to the amplification of proteins involved in the DDR signaling cascade. 20,24,25Cells lacking H2AX have been observed to exhibit slower repair processes and more chromosomal rearrangements than cells with H2AX present, highlighting the significant role played by gH2AX in maintaining genomic stability.However, despite its importance, the lack of standardized parameters and technical heterogeneity in quantifying gH2AX limits its utility as a biomarker for DNA damage or repair, and further research on gH2AX as a biomarker is necessary to establish its clinical application. 26o complementary repair mechanisms have evolved to coun- and post-replication in the S and G2 phases of the cell cycle when non-damaged repair templates are available.This process is considered error-free and preserves genomic information.The repair is initiated by the MRE11-RAD50-NBS1 (MRN) complex, which detects and binds to the DSB to bring the ends together.The serine/threonine kinase ATM (ataxia telangiectasia mutated) is recruited upon complex binding and phosphorylates key proteins, including γ-H2AX, which plays roles in DNA damage checkpoints and catalyzing repair.
The 5′-ends of the DSBs are then nucleolytically cleaved, creating 3′ overhangs.RAD51 generates a nucleoprotein that forms a D-loop conformation, connecting and synthesizing DNA strands. 23,24,27In contrast, NHEJ repairs DSBs by directly rejoining the two broken ends without using a homologous sequence as a template.During various cell cycle stages, NHEJ is the dominant repair mechanism, especially in G0 and G1 phases and during V(D)J recombination and immunoglobulin recombination in lymphocytes.In NHEJ repair, the two broken ends are rejoined through the action of the DNA-end binding protein Ku, which recruits DNA-dependent protein kinase catalytic subunits (DNA-PKcs).DNA-PKcs then recruit XRCC4 and ligase IV, facilitating the re-ligation of the broken ends.This repair process is faster but less accurate than HR.]27

| Natural sperm DNA repair mechanisms
Spermatozoa are remarkably resilient cells, enduring the challenging journey from the epithelial testes to the fallopian tubes to reach the oocyte.Mammalian sperm DNA structure is highly compacted and unlike any other eukaryotic cell.This structure is achieved through specialized sperm chromatin packaging, which maintains DNA integrity and organization from spermatogenesis until fertilization.The compaction process occurs during spermiogenesis, wherein histones are partially replaced by protamines-proteins rich in arginine and cysteine.The DNA-protamine complex structure involves protamines binding to the minor groove of DNA, neutralizing the backbone, forming hydrogen, hydrophobic, and disulfide bonds between each protamine at every DNA turn.9][30][31] However, despite this safeguarding mechanism, the DNA is still prone to damage in this compacted state. 32,33Protamine deficiencies show an inverse correlation with DNA fragmentation, 34 and insufficient nucleosome-to-protamine remodeling is associated with infertility. 35A replication is an integral process in all living organisms, yet DNA is highly vulnerable to damage.Damage can occur due to oxidative stress, spontaneous replication errors, and cellular metabolism, leading to the generation of as many as 10 5 lesions per cell per day.36 Several theories regarding the causes of sperm DNA damage have been proposed and include ROS damage, sperm chromatin package defects, and abortive apoptosis.Multiple studies have now identified a positive correlation between ROS levels and DNA fragmentation.37 While maintaining a baseline level of physiological ROS appears to decrease DNA fragmentation and be essential for oocyte-sperm fusion (capacitation), elevated ROS levels lead to higher rates of DNA fragmentation, showing an inverse correlation with semen volume, the percentage of motile spermatozoa, and sperm linearity.38,39 To counteract DNA damage in spermatozoa, five natural repair mechanisms have evolved.These mechanisms are nucleotide excision repair (NER), base excision repair (BER), DNA mismatch repair (MMR), DSB repair mechanisms, and post-replication repair (PRR).
NER primarily removes bulky lesions, such as pyrimidine or thymine dimers caused by UV exposure, as well as bulky adducts and DNA intra-strand cross links that distort the helix's structure.XPC/RAD23B proteins scan for damage detection in this process.On the contrary, BER is responsible for repairing non-helix distorting lesions, particularly those resulting from deamination, oxidation, and free radical damage.The BER mechanism involves DNA glycosylases that recognize and remove damaged bases by cleaving the N-glycosidic bond to deoxyribose.MMR functions as a proofreader during DNA replication, identifying base-base mismatches and insertion-deletion loops through antigen proteins MutS and MutL.This process significantly increases DNA replication stability by about 100-fold.Among various types of DNA damage, DSBs are the most harmful and can result from factors like ROS, failed DNA replication, repair, and recombination, meiosis, as well as exposure to IR or chemotherapeutic agents.
The repair pathways for DSBs include HR and NHEJ.HR primarily resolves DSBs during the S and G2 phases of the cell cycle, using the MRN complex comprising MRE11, RAD50, and Nijmegen breakage syndrome 1 (Nbs 1) proteins.1][42][43][44] PRR, working at the replication fork level, facilitates ongoing synthesis of damaged genomes.To prevent replication fork stalls, PRR employs trans-lesion synthesis (TLS) and template switching (TS).TLS involves specialized polymerases that replicate directly across lesions, whereas TS operates by utilizing genetic information from the newly synthesized chromatid as a template for replication, effectively avoiding the potential for fork-stalling lesions. 19Natural sperm DNA repair mechanisms are summarized in Figure 2.
Although there is a comprehensive list of repair mechanisms, spermatozoa possess a limited repair repertoire due to their condensed nature, which renders their DNA essentially inert to facilitate a safe union with the oocyte.Mature sperm face significant barriers to utilizing repair mechanisms, given their protamine-rich DNA and compaction level.During spermatogenesis, DNA damage repair occurs until the third week, after which sperm DNA undergoes significant condensation alongside the downregulation of repair mechanisms. 276][47] While a truncated BER pathway has been observed in spermatozoa through the presence of the enzyme OGG1, responsible for cleaving DNA base adducts, the primary responsibility for reducing the risk of paternally inherited genomic errors lies with the oocyte. 46The current accepted paradigm within sperm DNA repair mechanisms post-fertilization is that the oocyte plays a crucial role in rescuing the sperm's DNA damage.This reliance is supported by the presence of maternal mRNAs and proteins existing in the oocyte before ovulation and the high expression of DNA repair genes in human oocytes. 45,48,49he SCSA evaluates the susceptibility of sperm DNA to denaturation using acridine orange fluorescence to identify DSBs and SSBs. 53,54In contrast, the SCD assay relies on microscopic observation of a "halo," which represents chromatin dispersion following denaturation and deproteinization.Sperm with fragmented DNA do not exhibit this characteristic halo of dispersed DNA loops seen in non-fragmented DNA, making it useful for detecting both DSBs and SSBs. 51The Comet assay involves lysing cells under neutral or alkaline conditions to form deproteinized nuclei, and the resulting DNA is electrophoresed.Fragmented DNA moves toward the anode, creating a visual pattern resembling a comet, with the extent of the tail indicating the level of DNA damage.

| Type of SDF testing
6][57] Given the differential repair mechanisms for DSBs and SSBs, it is crucial to distinguish between these types of fragmentation in clinical applications related to ICSI or IVF ART.The 2T comet assay provides a solution to this problem by differentiating the total level of sperm DNA fragmentation (SDF) into SSBs and DSBs in individual cells. 53Tests that detect γH2AX, the phosphorylated form of the histone H2AX, immediately following DSBs, have emerged as promising predictors of success with ICSI, but further research involving randomized control trials is needed before they can be applied clinically. 27,58ile several tests are available for SDF testing, the lack of consistency in assay sensitivity and specificity for clinical applica- to identify clinical SDF cutoffs for predicting pregnancy, leading to variability in results. 50A review article on clinical guidelines for SDF compiled different studies that attempted to identify clinical cutoffs for predicting natural or ART pregnancies using these assays.The TUNEL assay showed cutoff values ranging from 4% to 20%, with most values falling in the late teens, while the Comet assay ranged from 44% to 56%.The SCSA results ranged from 10.4% to 30.3%, and the SCD assay ranged from 9.7% to 28.5%, with most values falling in the late 20s.Such differences among assays make it challenging to establish a universal SDF analysis standard.
Several studies have made claims about the predictive value of different assays for IVF and ICSI outcomes, but contradictory results have also been reported.However, there is a lack of comprehensive studies that directly compare the different assays, resulting in a lack of consensus regarding the standard threshold for defining increased DFI across these assays.This inconsistency poses challenges in interpreting the data and limits the universal clinical implementation of these assays for patients with infertility.For instance, a comparative study of the TUNEL, SCSA, and comet assays found weak correlations among the three when analyzed as continuous variables, making it questionable to take these results at face value. 54 ples experiencing RPL because sperm from patients with RPL may have higher SDF levels. 50The 6th Edition of the WHO manual for laboratory semen testing, published in 2021, includes a dedicated section to SDF and highlights how meta-analyses have suggested that SDF may affect embryo development, implantation, and natural and assisted pregnancies.The manual outlines the protocols for conducting the TUNEL assay, SCD test, Comet assay, and SCSA assay. 4The European Association of Urology's 2021 guidelines for sexual and reproductive health recommend performing SDF testing in the evaluation of couples experiencing RPL and unexplained infertility. 152][63] The complexity of infertility, which involves multiple contributing factors between couples, makes it challenging to attribute SDF as the sole reason for the need for ART.Therefore, incorporating SDF testing into clinical and professional health organization guidelines presents difficulties.

| Effect of SDF on fertility outcomes
Over the last two to three decades, numerous studies, including clinical trials, have been conducted to investigate the impact of SDF on fertility outcomes.Many systematic reviews and meta-analyses, particularly ART settings, have also been conducted to summarize its effects. 64Most of the meta-analyses have shown significant adverse effect of high SDF on fertility outcomes.However, the significance of SDF in clinical practice is still controversial and the evaluation of SDF is not recommended for routine initial evaluation of AMAB partners. 17This is mainly due to the studies analyzed by meta-analyses being cohort studies and lacked randomization.This truth results in weak connection of SDF on initial management of infertile couples.Oocytes are known to play an important role in repairing sperm DNA damage. 65Yet, its capacity depends on assigned female at birth (AFAB) individuals as the extent of repair mechanism contribution and the threshold of sperm DFI at which an oocyte can repair such damage are unclear.The oocytes' mechanistic involvement further contributes to complexity in evaluating the effect of SDF in infertility.In the fertilization and embryo development process, the paternal gene derived from spermatozoa will be activated after zygotic transcriptional activation, known as late paternal effect. 66The impact of high SDF on the reproductive process is more apparent after the 4-to 8-cell embryo stage rather than during fertilization.This is supported by studies showing that high SDF negatively affects embryo development but not fertilization outcomes. 67,68On the other hand, the effect of SDF on pregnancies or live births, both in natural pregnancies and ART settings, are more heterogeneous.
This heterogeneity is partly due to confounding AFAB factors that are challenging to adjust and account for in research settings.
5][76] Thus, the impact of SDF appears lower in studies of ICSI compared to those of IVF, despite the fact that they both needs in vitro culture and implantation of embryos.The reason for this is not fully understood.One possible reason is that gametes in ICSI have less exposure to the surrounding environment in prolonged culture as opposed to with IVF.This may result in less sperm DNA damage at the time of fertilization. 77The effect of SDF on live birth rates is more variable, with recent meta-analyses showing no significant relationship between high SDF and live birth rates, 76 while previous large-scale meta-analyses reported significantly reduced live birth rates in the high SDF group after both IVF and ICSI. 78

| Spontaneous pregnancy
Zini conducted a meta-analysis that included three studies [79][80][81] with a total of 616 cases.The analysis revealed a significant correlation between sperm DNA damage and the failure to achieve a natural pregnancy (odds ratio: 7.01, 95% confidence interval [CI] 3.68-13.36,p < 0.001). 69Although the effect of conventional parameters such as concentration or motility was not corrected in the meta-analysis, SDF was proven as an independent factor for infertility by multivariate regression or other analysis in included studies.However, there have been only limited studies conducted to investigate whether SDF can predict the likelihood of natural pregnancy.Additionally, the effect of SDF on live birth rate was not examined in this study.To obtain more robust evidence in the relation between SDF and these outcomes, large-scale prospective cohort studies designed with low risk of bias are needed.
Studying spontaneous pregnancies presents challenges due to the high number of confounding factors that can influence the outcomes.

Fertilization and embryo quality
Regarding the fertilization rate, Chen et al. conducted a meta-analysis that included 12 articles. 84They found that there was no statistically significant association between high SDF and fertilization rate, both in IVF (RR = 0.94, 95% CI 0.77-1.14,p = 0.61) and ICSI (RR = 0.79, 95% CI 0.52-1.18,p = 0.25).Li et al. also reported similar results, showing no significant association between high SDF and fertilization rate in both IVF and ICSI. 70The association between SDF and embryo quality has been the subject of several meta-analyses and reviews. 72,74,85mon et al. reviewed 80 articles comprising 14 871 treatment cycles 85 and found that 27 out of 80 (34%) studies reported a significant association between higher SDF and lower embryo quality.2 This suggests that high SDF is indeed associated with lower embryo quality, and the lack of significant association between SDF and fertilization rates may be attributed to the late paternal effect.

Implantation rate/ clinical pregnancy rate
Ribas-Maynou et al. conducted a meta-analysis and reported that high SDF had a negative effect on IVF, with a significant impact on implantation (RR = 0.68; 0.52-0.89;p < 0.01) and pregnancy rates (RR = 0.72, 95%CI:0.55-0.95,p = 0.02).However, the latter effect turned out to be not statistically significant after adjusting for publication bias.In contrast, implantation and pregnancy rates with ICSI were not significantly affected by SDF. 762 However, the pregnancy rate after ICSI has been shown to have an insignificant association with SDF in most meta-analyses. 69,70,72,73,75,76Only the meta-analysis conducted by Simon showed a statistically significant impact of SDF on clinical pregnancy after ICSI, 73 while the other five meta-analyses have concluded that the association was statistically insignificant.
This difference in the impact of SDF between IVF and ICSI may be attributed to the difference in their procedures.In ICSI, spermatozoa are manually injected into the oocyte, bypassing the random fertilization process.Conversely, in IVF, spermatozoa are co-incubated with oocytes, and fertilization occurs randomly as spermatozoa penetrate the zona of the oocyte.During this process, natural sperm selection can occur, where spermatozoa with high SDF might be naturally excluded.In IVF, however, the gametes are subjected to prolonged culture during the fertilization process.Thus, spermatozoa may be damaged by the surrounding environment such as ROS in culture media, Conversely, spermatozoa are injected into the oocyte within a few hours of ejaculation in ICSI and they can avoid the exposure. 86Another possibility is that bias between patient populations seeking these ARTs.Patients undergoing ICSI tend to have worse clinical conditions that are independent of SDF.It is difficult to eliminate all these bias factors, even if adjustment analyses are incorporated.

Live birth rate
Although the most important outcome for infertile couples is live birth rate, only a few studies have evaluated this as an outcome, and each study had a relatively small sample size.Two available metaanalyses have evaluated live birth rates. 76,78 Similarly, Deng et al. also found no significant effect of SDF on live birth rates after combined IVF and ICSI. 72

| Recurrent pregnancy loss
RPL has posed significant challenges in the field of reproductive medicine, and while various factors are believed to contribute to its occurrence, the underlying mechanism is not fully understood. 87 addition to the well-studied maternal causes, there is a growing body of evidence highlighting the impact of AMAB factors, including SDF.Recent animal studies have demonstrated that sperm DNA damage can lead to the fragmentation and random distribution of paternal chromosome segments in developing embryos, resulting in chaotic mosaicism with non-identical genotypes in cells. 88though the oocyte can repair some sperm DNA damage after fertilization, high levels of damage beyond the oocyte's repair capacity can lead to poor embryo development, implantation failure, and miscarriage. 47,89In a meta-analysis conducted by Robinson et al. in 2012, involving 2969 couples, it was demonstrated that SDF is associated with sporadic miscarriage (RR = 2.16, 95%CI: 1.54-3.03,p < 0.00001). 90In the context of RPL, a recent meta-analysis by McQueen et al. also showed that the partners of patients with a history of RPL have a significantly higher rate of SDF compared to the partners of fertile control patients without RPL (mean difference of 10.7%, 95% CI: 5.82-15.58,p < 0.0001). 91Considering the high risk of miscarriage associated with abnormal SDF, the latest ASRM guidelines now recommend evaluating SDF in addition to karyotype analysis for couples experiencing RPL. 17 Similarly, the 2022 ESHRE guideline for RPL also acknowledges the importance of assessing SDF in such couples for diagnostic purposes, not solely for explanatory purposes, based on the emerging evidence from recent analyses. 87he compiled meta-analyses on SDF based on the type of conception and their effects are presented in Table 2. Overall, previous studies have shown the potential of SDF as a predictor of reproductive outcomes, particularly in ART.Evaluating SDF may aid clinicians in making decisions about whether to proceed with ICSI for patients with high SDF, where the impact of SDF on outcomes is minimal or insignificant.However, it should be noted that the role of SDF as a predictor in reproductive outcomes is still not definitively established.Some original studies have reported no significant association between SDF and reproductive outcomes. 92,93The overall impact of meta-analyses is also limited by the scarcity of well-conducted RCTs in previous studies.As discussed earlier, patient cohorts in clinical conditions may introduce biases, and the number of non-adjustable confounding factors increases when evaluating final outcomes such as live birth rates.In addition, there is heterogeneity in the assays used for SDF measurement and the determination of cutoff values.Thus, standardizing the assays and cutoff values is crucial for establishing SDF as a more robust marker in future clinical practice.

| SDF and cost
The estimated cost of each SDF testing kit ranges between $175-400 in the United States. 94SDF testing is usually not covered by medical insurance alongside other fertility-related therapeutic interventions. 95In a case scenario with high SDF, it will cost more than $1000 for the SDF testing.Additional costs for main treatment to improve SDF include medication and varicocelectomy.
Moreover, equipment and running cost also need to be considered

| Lifestyle changes
High levels of SDF have been associated with various lifestyle factors that may negatively impact overall health.These include metabolic conditions such as diabetes mellitus, obesity, and tobacco smoking.A recent meta-analysis by Szabo et al. revealed that impaired glycemic control and smoking are associated with higher levels of SDF. 13 This suggests that adopting a healthy lifestyle, which includes a balanced diet, regular exercise, and refraining from smoking, may help reduce or prevent high levels of SDF. 96Specifically, engaging in high-intensity exercises, following a diet rich in vegetables, fish, seafood, and whole grains (e.g., the Mediterranean diet), and reducing alcohol intake may have favorable effects on SDF. 97Cigarette smoking in particular has been strongly associated with high SDF.Studies have consistently demonstrated that smokers tend to have significantly higher levels of SDF when compared to non-smokers. 97,98The exact mechanism behind this association is likely complex and involves factors such as higher levels of ROS and apoptotic caspases in infertile smokers.
0][101] Overall, these studies emphasize the importance of quitting or reducing cigarette smoking to prevent higher SDF levels, which could have a significant impact on fertility and overall reproductive health.

| Varicocele repair
Varicocele is the second most common cause of AMAB infertility after idiopathic infertility, affecting approximately 35%-44% of infertile AMAB patients. 102The condition is characterized by reflux in the spermatic vein, leading to increased temperature in scrotum, which in turn degrades spermatogenesis.Moreover, the testicular microenvironment experiences reduced oxygen levels due to the inefficient venous return, resulting in heat stress and oxidative stress that adversely affect AMAB fertility and contribute to SDF. 103 As a result, patients with clinical varicocele tend to have higher SDF levels compared to controls. 104Fortunately, varicocele repair has been found to significantly reduce SDF levels in patients with clinical varicocele, as reported in recent metaanalyses. 105,106Moreover, studies have indicated that varicocele repair in infertile patients with clinical varicocele and high SDF can lead to significant improvements in clinical pregnancy and live birth rate after ICSI. 107Thus, varicocele repair is recommended for infertile AMAB patients with clinical varicocele and high SDF.
However, there has been some debate about whether varicocele repair should be offered to patients with otherwise normal semen parameters but high SDF. 108A recent global survey on varicocele management showed that 34.7% of reproductive experts consider varicocele repair for patients with a history of infertility and increased SDF, even if conventional semen parameters are normal.
Nonetheless, further well-designed studies are needed to establish the efficacy of varicocele repair in such cases. 109

| Role of antioxidants
The potential of antioxidant treatment to mitigate the deleterious impact of ROS on SDF has been explored as a promising approach TA B L E 2 Literature of meta-analyses on sperm DNA fragmentation and reproductive outcomes.has been studied in various centers and has been found to be independently associated with improvements in SDF according to multivariate analysis. 110,111However, the results may be limited by the fact that some studies were conducted at single centers or retrospective in nature.Meta-analyses of RCTs have demonstrated a significant reduction in SDF compared to a placebo, 14 specifically with the use of butylated hydroxytoluene (BHT), vitamin E, and tempol. 112Nonetheless, the optimal dosing, regimen length, and timing for antioxidant therapy have not been fully established, and certain studies have suggested no significant difference in SDF for otherwise healthy, AMAB infertile patients. 113The actual impact of antioxidant supplementation for AMAB subfertility on pregnancy rates and live birth has been a big controversial topic.Although the latest Cochrane Review has shown that antioxidant supplementation may increase clinical pregnancy rates (OR = 1.89) and change of live birth (OR = 1.43), the analysis has concluded that the overall certainty of evidence was very low from small number of RCTs. 114e Males, Antioxidants, and Infertility (MOXI) randomized controlled trial as a well-known RCT demonstrated that plasma antioxidant levels at baseline were not predictive of successful pregnancy or live birth. 115Additionally, antioxidant levels no statistically TA B L E 2 (Continued) significant correlation between-at baseline or following 3 months of treatment-antioxidant levels (specifically vitamin E, selenium, and zinc) and SDF. 115Although MOXI trial is an important RCT in this topic, there is argument because of inherent shortcomings in the study design.There is a criticism that this study was ended after the pilot phase of 3 months completed before it reached decent power size. 116Larger RCT with robust methodology needs to be conducted to provide further conclusions.Overall, oral antioxidant therapy may be a cost-effective option for patients with elevated SDF; however, the selection of treatment should be guided by individual decisions made between the healthcare professional and the patient.It is also essential to consider any potential adverse effects of supplementation.It is crucial to note that antioxidants are not approved by the government as a standard treatment for infertility.

| Role of hormonal versus non-hormonal therapy
Studies have shown that AMAB infertility is associated with lower serum testosterone levels and reduced testosterone-to-estradiol ratios. 117As a result, several studies have focused on therapies that can modulate the hypothalamic-pituitary-gonadal axis and/or the peripheral conversion of hormones to explore their impact on SDF.Some evidence suggests that letrozole (a peripheral aromatase inhibitor) or direct administration of follicle-stimulating hormone (FSH) can significantly reduce SDF and contribute to successful intrauterine pregnancy. 117,118Age, baseline testosterone levels, and testicular volume may be predictors of response to FSH administration. 117,118Regarding treatment regimens, there is currently no definitive evidence to guide specific paradigms.However, some data suggests that sequential treatment with human chorionic gonadotropin (hCG) and FSH could be effective due to the luteinizing hormone (LH)-like effect of hCG. 119Antioxidant therapy, as discussed earlier, is a non-hormonal approach for managing elevated SDF.In addition, evidence suggests that addressing infectious diseases may play a crucial role in reducing high SDF.For instance, combination antiretroviral therapy has been found to decrease SDF in HIV patients, and AMAB individuals with human papillomavirus (HPV) infections tend to have higher levels of SDF. 120,121This suggests that timely vaccinations and standard interventions to reduce viral load could be beneficial in reducing or preventing high SDF.

| Testicular versus ejaculate sperm
Testicular sperm has been found to have lower levels of oxidative damage, potentially leading to decreased SDF and increased fertility rates compared to ejaculated sperm. 122However, SDF testing in testicular sperm lacks standardization.Obtaining testicular sperm also requires surgical intervention, which carries some common surgical risks (e.g., surgical site infection, testicular swelling, damage to surrounding structures, etc.), although these complications are rare. 122,123Despite these risks, critical analyses of meta-analyses and primary studies comparing testicular and ejaculated sperm have demonstrated higher pregnancy and live birth rates in patients with high SDF using testicular sperm. 124,125Robust RCTs are still lacking, leading the European Association of Urology to refrain from recommending routine testicular sperm retrieval for infertile AMAB patients with high SDF without a full explanation of the risks and the lack of current RCT-derived data to patients. 125Interestingly, a recent survey among global sexual health experts has revealed that a significant portion of urologists might recommend testicular sperm retrieval in select cases. 126Thus, the recommendations regarding testicular sperm retrieval for AMAB patients with high SDF remain controversial and unresolved.

| Short abstinence interval
Although WHO manual recommends 2-7 days of abstinence for semen analysis to standardize the methodology, an even shorter abstinence interval is reported to be effective in improving semen parameters and SDF. 127In the absence of ejaculation, spermatozoa accumulate in the epididymis.In the abstinence interval, spermatozoa is exposed to the damaging of ROS generated by abnormal spermatozoa and granulocytes which results in high SDF. 128The mechanism of improvement in semen quality in second ejaculation has not been fully unveiled, but it is hypothesized that shorter epididymal transit time can reduce exposure to ROS, and also cause biochemical changes in consecutive ejaculations, which results in reducing sperm damage. 129Although current guidelines do not state the efficacy of shorter abstinence, recent systematic meta-analysis has shown that a shorter abstinence interval was associated with better semen motility and morphology, and lower SDF. 130A systematic review by Ayad et al. showed that shorter abstinence decreased the semen volume and concentration while it increased sperm motility significantly. 131Studies have also been focusing on very short abstinence which means second ejaculation less than 3 h after the first on the same day.A study in Italy compared semen parameters from normozoospermic and oligoasthenoteratozoospermia (OAT) AMAB individuals after a 2-to-7-day abstinence period and a one-hour abstinence period.Both groups revealed a significant improvement in normal morphology and a decrease in SDF with a shorter abstinence period, and only OAT group demonstrated an improvement in motility. 132Recent meta-analysis from the same group conducted by Barbagallo et al.
showed that sperm concentration and motility were significantly increased in second ejaculate of OAT patients.In contrast, semen volume and SDF was significantly decreased in the second ejaculate. 133Especially in ART settings, the sperm quality is more important than the number of spermatozoa as infertile couples do not need so much total sperm count as needed in spontaneous pregnancy or IUI.Therefore, these findings may suggest that OAT patients undergoing ART may have more benefit from a very short abstinence period.Overall, short abstinence interval seems a simple and useful strategy for obtaining spermatozoa with lower SDF and better quality.More robust RCTs are needed to establish strong recommendations.another are currently lacking. 126The management flowchart for cases with high sperm DNA fragmentation is shown in Figure 3.

| CON CLUS ION
This narrative review has presented the latest research articles covering various aspects of SDF, including its mechanism, sperm DNA repair, evidence regarding its impact on reproductive outcomes, and its management.While the efficacy of SDF as a marker has been demonstrated, its application is currently limited to specific situations.For decades, many sperm functional tests have been

ACK N OWLED G M ENTS
None.

CO N FLI C T O F I NTE R E S T S TATE M E NT
Authors declare no Conflict of Interests for this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author, SK, upon reasonable request.

H U M A N/A N I M A L R I G HTS S TATEM ENT
This article does not contain any studies involving human or animal subjects.

Figure 1 .
Figure 1.SSBs are the most prevalent DNA lesions and can arise from various sources such as ionizing radiation (IR), oxidative stress, intracellular or environmental genotoxins, or intermediates of normal DNA metabolic processes.These breaks are detected by poly (ADPribose) polymerase 1 (PARP1) and 2 (PARP2), leading to enzymatic activation and the synthesis of poly (ADP-ribose) (PAR) chains.
teract the damaging effects of DSBs: homologous recombination (HR) and non-homologous end-joining (NHEJ).The choice between these repair pathways depends on the cell cycle stage and the extent of DNA breakage.HR primarily operates during DNA replication F I G U R E 1 Summary of various types and underlying molecular mechanisms of sperm DNA damage.
tions, along with discrepancies in the literature regarding the overall association between increased SDF and clinical fertility outcomes among different assays, complicate the universal predictive abilities of these tests.The quest to establish a consistent relationship between SDF and fertility outcomes faces challenges due to the lack of a standardized definition and assay to define increased DNA fragmentation index (DFI) in couples experiencing RPL caused by sperm-related factors.Each assay has its own threshold values A study by Ribas-Mayou et al. compared the TUNEL, SCSA, SCD, and different comet assay types within a single population.The resultsshowed statistically significant differences in SDF between fertile and infertile patients using the TUNEL assay, SCSA, SCD test, and alkaline comet assays.However, no differences were found between patients when using the neutral comet assay.Correlation analyses indicated the strongest correlations between the cytometric assays (TUNEL and SCSA), as well as correlations between the SCD test and SCSA, and the SCD test and TUNEL assay.These findings further highlight the complexity in establishing a clear consensus on the parameters for elevated DFI within SDF assessment.The correlation values were relatively lower between the alkaline comet assay and the SCD test, the alkaline comet and SCSA, and the alkaline comet and TUNEL assay, with no significant correlations between the neutral comet assay and the other four methodologies.To assess the clinical utility of different SDF tests for predicting infertility, Ribas-Maynou et al. also utilized receiver operating characteristic curves to determine sensitivity and specificity.Their results showed the alkaline comet assay as the most predictive method, followed by the TUNEL assay, the SCD test, SCSA, and the neutral comet assay.This study argues that different techniques may measure various aspects of SDF, which supports the claim that SA protocols should incorporate multiple SDF testing methods.51The debate surrounding the clinical implementation of SDF testing is controversial among physicians and professional health organization guidelines.In 2013, the American Society for Reproductive Medicine (ASRM) guideline on the utility of sperm DNA integrity testing concluded that SDF is common in patients experiencing infertility, but the current testing methods lack reliable predictability for routine clinical use.59The ASRM's 2015 guideline for the diagnostic workup of infertile patients acknowledged SDF and its association with spontaneous recurrent miscarriage but highlighted the limited data available to support routine clinical inclusion of abnormal DNA-integrity assessments in the diagnostic process.The guideline addressed the clinical implications of abnormal SDF on IVF or ICSI outcomes.It emphasized that the available data on the relationship between abnormal DNA integrity and reproductive outcomes are limited, making routine testing currently impractical.The guideline called for further research and clinical studies to identify subgroups of patients who might benefit from SDF testing.60In contrast, the 2020 American Urological Association (AUA)/ASRM guidelines for diagnosing and treating infertile patients did not recommend SDF testing as part of the initial evaluation due to inadequate data to support routine usage.Although the guidelines acknowledged the negative association of DNA fragmentation with pregnancy rates and its positive association with miscarriages, they mentioned that the definition of high levels of SDF remains unclear.Medical associations advised counseling infertile patients about the potential association of high SDF with miscarriage and considered SDF testing for cou- by providers.Some of the assays such as TUNEL and SCSA need flow cytometry, which remains one of the most expensive pieces of laboratory equipment.The cost varies, but it typically ranges from $100 000-$500 000 in the United States.Running flow cytometry requires well-trained technicians, thus an additional barrier and cost.Although the cost of SDF testing is currently one of the important factors limiting its routine use in clinical practice, appropriate performance of SDF testing should ideally reduce the overall treatment cost by assisting robust clinical fertility decisionmaking process.

3. 6 . 7 |
Sperm selection in high SDFFinally, various methods for sperm selection have been explored to improve infertility rates in AMAB patients with high SDF undergoing assisted reproductive techniques.Initial studies on intracytoplasmic morphologically selected sperm injection (IMSI), which involves selecting sperm based on high magnification analysis of sperm morphology, did not show significantly improved clinical outcomes compared to ICSI.134However, later RCTs comparing common sperm selection techniques, such as density gradient centrifugation (DGC), physiologic ICSI (PICSI), and magnetic-activated cell sorting (MACS), revealed that combining PICSI and MACS with DGC led to significantly higher implementation and pregnancy rates compared to using DGC alone.135Briefly, PICSI involves placing sperm on hyaluronan microdots to mimic high DNA-integrity binding, leading to the selection of high-integrity sperm, whereas traditional ICSI relies only on morphology and motility for sperm selection.Conversely, MACS separates apoptosed sperm cells by recognizing externalized phosphatidylserine residues.135,136MACS may be particularly suitable for certain patient populations, as a study by González-Ravina and colleagues demonstrated comparable gestation, live birth, or miscarriage rates between MACs after DGC and DGC alone for couples undergoing donor IUI (D-IUI).136Although larger sample sizes and additional multicenter studies are necessary to determine the most effective sperm selection treatments for individuals with SDF, healthcare professionals can discuss selection techniques with patients while acknowledging that robust studies demonstrating the superiority of one technique over

F I G U R E 3
Flowchart illustrating the management for cases with high sperm DNA fragmentation.established in the research field.Within the field of AMAB factor infertility, conventional SA has been the standard routine marker in clinical practice, but it has its limitations.These include poor repeatability and inability to fully assess the fertility of patients.Therefore, there is a pressing need for more reliable markers to evaluate sperm quality, and SDF shows promise as a potential marker for the future.At present, global guidelines recommend SDF evaluation for couples with idiopathic AMAB factors and RPL.High SDF has been associated with lower pregnancy rates in IVF, but its impact on ICSI outcomes remains less clear.In couples experiencing recurrent failure in ART with the AMAB partner showing high SDF, testicular sperm extraction could be considered as an option.For varicocele patients with a history of infertility and normal semen parameters, SDF can help determine the application of varicocelectomy.To establish the true efficacy of SDF in real-world scenarios, higher-quality clinical trials, especially RCTs, are necessary.Addressing the variations in cutoff values and subjectivity in SDF assays is crucial for its broader application.Artificial intelligence (AI) could play a significant role in this regard, as recent studies combining SDF techniques with AI have shown promising results. 137,138Such attempts may reduce human effort and minimize the subjectivity and inter-variability in assays, which have been obstacles in determining the efficacy of SDF.In conclusion, despite its limitations, SDF holds potential for greater inclusion in clinical guidelines.High-quality clinical trials are essential for standardizing the choice of SDF assays and determine the appropriate DFI cutoff values.Additionally, advancements in assays, including improvements in complexity, subjectivity, and cost-effectiveness will enhance accessibility to SDF testing.While SDF is perhaps the best-studied nontraditional sperm test currently available, even this robust set of literature remains contentious, and SDF likely plays a modest role only for a subset of AMAB individuals with infertility.Nevertheless, SDF represents an excellent paradigm and can certainly act as a proof-of-principle for the field of andrology to explore other novel sperm tests and expand our knowledge of the diagnosis and treatment of idiopathic AMAB infertility.